Infiltration, accumulation, and survival of chimeric antigen receptor T (CAR-T) cells in solid tumors is crucial for tumor clearance. We engineered CAR-T cells to express interleukin (IL)-7 and CCL19 (7 × 19 CAR-T cells), as these factors are essential for the maintenance of T-cell zones in lymphoid organs. In mice, 7 × 19 CAR-T cells achieved complete regression of pre-established solid tumors and prolonged mouse survival, with superior anti-tumor activity compared to conventional CAR-T cells. Histopathological analyses showed increased infiltration of dendritic cells (DC) and T cells into tumor tissues following 7 × 19 CAR-T cell therapy. Depletion of recipient T cells before 7 × 19 CAR-T cell administration dampened the therapeutic effects of 7 × 19 CAR-T cell treatment, suggesting that CAR-T cells and recipient immune cells collaborated to exert anti-tumor activity. Following treatment of mice with 7 × 19 CAR-T cells, both recipient conventional T cells and administered CAR-T cells generated memory responses against tumors.

T-cell tolerance is the central program that prevents harmful immune responses against self-antigens, in which inhibitory PD-1 signal given by B7-H1 interaction plays an important role. Recent studies demonstrated that B7-H1 binds CD80 besides PD-1, and B7-H1/CD80 interaction also delivers inhibitory signals in T cells. However, a role of B7-H1/CD80 signals in regulation of T-cell tolerance has yet to be explored. We report here that attenuation of B7-H1/CD80 signals by treatment with anti-B7-H1 monoclonal antibody, which specifically blocks B7-H1/CD80 but not B7-H1/PD-1, enhanced T-cell expansion and prevented T-cell anergy induction. In addition, B7-H1/CD80 blockade restored Ag responsiveness in the previously anergized T cells. Experiments using B7-H1 or CD80-deficient T cells indicated that an inhibitory signal through CD80, but not B7-H1, on T cells is responsible in part for these effects. Consistently, CD80 expression was detected on anergic T cells and further up-regulated when they were reexposed to the antigen (Ag). Finally, blockade of B7-H1/CD80 interaction prevented oral tolerance induction and restored T-cell responsiveness to Ag previously tolerized by oral administration. Taken together, our findings demonstrate that the B7-H1/CD80 pathway is a crucial regulator in the induction and maintenance of T-cell tolerance. (Blood. 2010;116(8): 1291-1298) Introduction B7-H1 (CD274, PD-L1), a transmembrane glycoprotein belonging to immunoglobulin (Ig) superfamily molecule, plays an integral role in the regulation of immune tolerance and homeostasis. 1 Mice deficient of B7-H1 gene or wild-type mice treated with anti-B7-H1 blocking monoclonal antibody (mAb) exhibit exacerbated autoimmune phenotypes associated with an activation of self-reactive CD4 ϩ and CD8 ϩ T cells. [2][3][4][5] Tolerogenic functions of B7-H1 are dependent on its expression on hematopoietic or parenchymal cells, and mediated by its interaction with PD-1 receptor. 6-8 PD-1 is inducibly expressed on T cells after activation and delivers coinhibitory signals via immunoreceptor tyrosine-based switch motif in the cytoplasmic domain. 9,10 PD-1 signal interferes with phosphatidylinositol-3-kinase (PI3K) activity and subsequently inhibits interleukin-2 (IL-2) production, which eventually renders T cells anergic. 11 The mice deficient of PD-1 gene spontaneously develop autoimmune phenotypes, and single nucleotide polymorphisms of human PD-1 gene are associated with an increased risk of autoimmune diseases. [12][13][14][15][16] Recent studies by Butte et al discovered that B7-H1 interacts with CD80 (B7-1) in addition to PD-1. 17,18 In vitro studies using CD4ϩ T cells deficient of PD-1, CD28, and/or CTLA-4 indicated that B7-H1/CD80 interaction delivers bidirectional inhibitory signals to T cells. 17 These findings are consistent with previous observations implicating the presence of non-PD-1 receptor(s) of B7-H1. For instance, when the B7-H1/PD-1 interaction is blocked in models of T-cell tolerance, the effects of anti-B7-H1 antagonistic mAb in rest...

Purpose: To develop an adaptable gene-based vector that will confer immune cell specificity to various cancer types.Experimental Design: Human and mouse T cells were genetically engineered to express a chimeric antigen receptor (CAR) that binds a fluorescein isothiocyanate (FITC) molecule, termed anti-FITC CAR T cells. Various antibodies (Ab) currently in clinical use including cetuximab (Ctx), trastuzumab (Her2), and rituximab (Rtx) were conjugated with FITC and tested for their ability to bind tumor cells, activate T cells, and induce antitumor effects in vitro and in vivo.

Chronic graft-versus-host disease (GVHD) is the most common cause of poor long-term outcomes after allogeneic bone marrow transplantation (BMT), but the patho-physiology of chronic GVHD still remains poorly understood. We tested the hypothesis that the impaired thymic negative selection of the recipients will permit the emergence of pathogenic T cells that cause chronic GVHD. Lethally irradiated C3H/HeN (H-2 k) recipients were reconstituted with T-cell-depleted bone marrow cells from major histocompatibility complex [MHC] class II-deficient (H2-Ab1 /) B6 (H-2 b) mice. These mice developed diseases that showed all of the clinical and histopathological features of human chronic GVHD. Thymectomy prevented chronic GVHD, thus confirming the causal association of the thymus. CD4 T cells isolated from chronic GVHD mice were primarily donor reactive, and adoptive transfer of CD4 T cells generated in these mice caused chronic GVHD in C3H/ HeN mice in the presence of B6-derived antigen-presenting cells. Our results demonstrate for the first time that T cells that escape from negative thymic selection could cause chronic GVHD after alloge-neic BMT. These results also suggest that self-reactivity of donor T cells plays a role in this chronic GVHD, and improvement in the thymic function may have a potential to decrease chronic GVHD. (Blood. Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is considered to be an effective therapy for a variety of malignant and nonmalignant diseases of the lymphohematopoietic system. Chronic graft-versus-host disease (GVHD) is a complex multiorgan disorder with features of autoimmunity and immunodeficiency. 1,2 Chronic GVHD remains the major cause of late death and morbidity after allogeneic HSCT. 3,4 Unfortunately, despite success in the prevention of acute GVHD by the introduction of cyclosporine, the incidence of chronic GVHD has not decreased. 5-10 Its incidence is increasing with the greater use of unrelated or HLA-mismatched donors, older donors and recipients, donor lymphocyte infusions, and peripheral blood stem cell transplantation. These observations suggest that chronic GVHD is not simply a continuation of acute GVHD, and therefore novel strategies are required to prevent chronic GVHD. Although progress has been made in understanding the pathophysiology of acute GVHD, the basic pathophysiol-ogy of chronic GVHD remains poorly understood. The existing experimental models of chronic GVHD have certain limitations. For example, the most commonly used mouse model of chronic GVHD is induced by the injection of parental (P) cells into nonirradiated F1 recipients. 11 This model mimics sys-temic lupus erythematosus (SLE) with B-cell expansion, spleno-megaly, autoantibody production, and glomerulonephritis as a result of a cognate interaction between donor CD4 T cells and host B cells. 12,13 Another murine model, B10.D2 (H-2 d) into an irradiated BALB/c (H-2 d) model, mimics human chronic GVHD with fibrosis of the skin and exocrine glands and hepatic and pulmonary involve...

IntroductionSoluble immune aggregates bearing intact Fc fragments are effective treatment for a variety of autoimmune disorders in mice. The better to understand the mechanisms by which Fc-bearing immune complexes suppress autoimmunity, and to develop a platform for clinical translation, we created a series of fully recombinant forms of polyvalent IgG2a Fc, termed stradomers, and tested their efficacy in a therapeutic model of collagen-induced arthritis (CIA) and preventive models of both idiopathic thrombocytopenic purpura (ITP) and graft-versus-host disease (GVHD).MethodsStradomers were created by engineering either the human IgG2 hinge sequence (IgG2H) or the isoleucine zipper (ILZ) onto either the carboxy or amino termini of murine IgG2a Fc. Multimerization and binding to the canonical Fc receptors and the C-type lectin SIGN-RI were evaluated by using sodium dodecylsulfate-polymerase chain reaction (SDS-PAGE) and Biacore/Octet assays. The efficacy of stradomers in alleviating CIA and preventing ITP and GVHD was compared with "gold standard" therapies, including prednisolone and intravenous immune globulin (IVIG).ResultsStradomers exist as both homodimeric and highly ordered sequential multimers. Higher-order multimers demonstrate increasingly stable associations with the canonic Fcγ receptors (FcγRs), and SIGN-R1, and are more effective than Fc homodimers in treating CIA. Furthermore, stradomers confer partial protection against platelet loss in a murine model ITP, but do not prevent GVHD.ConclusionThese data suggest that fully human stradomers might serve as valuable tools for the treatment of selected autoimmune disorders and as reagents to study the function of Fc:FcR interactions in vivo.

B and T lymphocyte attenuator (BTLA) is a coinhibitory receptor that interacts with herpesvirus entry mediator (HVEM), and this interaction regulates pathogenesis in various immunologic diseases. In graftversus-host disease (GVHD), IntroductionActivation of T lymphocytes is regulated by 2 distinct signals: one is a primary signal delivered by T-cell receptor interaction with antigenic peptide/major histocompatibility complex (MHC), and the other is a cosignal delivered by interactions between cosignal receptors on T cells and their ligands on antigen-presenting cells. 1,2 Cosignaling receptors transmit stimulatory or inhibitory signals according to characteristics of their intracellular signaling motifs, and a balance of cosignals defines the fate of T-cell responses (ie, optimal activation or deactivation/tolerance induction). 3,4 Approaches to regulate cosignaling functions have been applied as novel and promising immunotherapies in various disorders, including cancer, infectious diseases, autoimmunity, organ transplantation, and graft-versus-host disease (GVHD).B and T lymphocyte attenuator (BTLA) is a cosignaling molecule that structurally belongs to the immunoglobulin (Ig) superfamily, expressed on broad ranges of immune cells, including T cells, B cells, and dendritic cells (DCs). [5][6][7] Intracellular domain of BTLA has 2 immunoreceptor tyrosine-based inhibition motifs, to which SH2 domain-containing protein tyrosine phosphatase-1 and tyrosine phosphatase-2 are recruited. 5,8,9 This signaling characteristic is consistent with its immune inhibitory functions, as BTLA gene-deficient mice exhibit an enhanced susceptibility to autoimmune diseases and increased inflammatory responses. 5,10-14 BTLA coinhibitory signal is induced by interaction with its endogenous ligand herpesvirus entry mediator (HVEM), a member of tumor necrosis factor-receptor superfamily. 8,15 In addition to BTLA, HVEM has 3 other binding partners, LIGHT (lymphotoxin-like, inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), CD160 and lymphotoxin-␣. 16 LIGHT-HVEM interaction transmits HVEM-positive cosignal into T cells via activation of nuclear factor-B (NF-B) signaling pathway. [16][17][18] HVEM interactions with BTLA and LIGHT are dependent on distinct extracellular regions of HVEM (ie, cysteine-rich domain-1 for BTLA while opposing cysteine-rich domain-2 and -3 sites for LIGHT binding), and it has been suggested that ternary LIGHT-HVEM-BTLA complex either augments or disrupts HVEM-BTLA interactions according to soluble or membrane form of LIGHT. 19 In contrast to negative cosignaling functions of BTLA, recent studies also suggested prosurvival effects of BTLA. For instance, in nonirradiated parent-into-F1 GVHD model, transfer of BTLAknockout (KO) donor T cells resulted in significantly diminished donor-antihost responses because of an impaired donor T-cell survival. 20 In addition, intestinal inflammation induced by a transfer of BTLA-KO T cells into Rag-KO mice was ...

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FTY720 is a novel immunosuppressant that improves the outcomes after solid organ and bone marrow transplantation (BMT) due to the sequestration of T cells into LN. We tested the hypothesis that the sequestration of donor T cells in LN by FTY720 would enhance their interaction with host APC, thus causing a greater degree of activationinduced apoptosis of alloreactive T cells, and thereby resulting in a reduction of graftvs.-host disease (GVHD). The short-term administration of FTY720 improved the recipient survival after allogeneic BMT. FTY720 treatment facilitated a rapid contraction of the donor T cell pool in association with an increased degree of apoptosis of donor T cells. The donor T cell reactivity to host alloantigens was diminished in host's LN and adoptive transfer of donor T cells isolated from LN of FTY720-treated recipients of allogeneic BMT induced less severe GVHD in secondary recipients than the transfer from controls. Caspase-dependent apoptosis was involved in this mechanism because FTY720-induced protection was abrogated when a pan-caspase inhibitor was administered. These findings thus demonstrate the presence of a novel mechanism by which FTY720 modulates the allogeneic T cell responses: namely, by the induction of activation-induced apoptosis of alloreactive T cells in LN.